User manual - Check
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1. Check MDR e CT 103 B The amount of bacterial DNA added in step A was probably insufficient the DNA extraction may have failed or the DNA concentration may have been too low Please repeat the test with a new DNA extract The software indicates ESBL suspected The software did not find sufficient spots to give a conclusive result Please inspect the picture visually an air bubble or dust particles may interfere with the result Tap the AT gently or pipette the liquid gently up and down and retry to take the image Please repeat the test if the results do not change The software indicates Picture not found or Image capture error Check if the Check Points Tube Reader is properly installed Please contact Check Points Technical Support at support check points com if reinstallation of the reader does not solve this problem The AT image is covered with small spots There may be various causes for this phenomenon Most likely the array dried out during the detection step Please make sure that the AT always contains sufficient amounts of reagents Detection Buffer Blocking Buffer conjugate dilution or Staining Solution This is particularly critical during the incubation steps at 50 C In most cases the software will be able to handle these small spots If not repeat the detection step with a new AT The AT was incubated for more than 15 minutes with Staining Solution before taking the image Results may be unreliable due t2. 7 Transfer 10 ul reaction mixture from each reaction tube to the corresponding Please note that one reaction tube corresponds to one AT The total volume of the AT will be 310 ul The lid of the AT should be labelled for reference Note e Samples may contain a white colored precipitate This is due to denaturation of one of the reaction components a protein stabilizer The presence of this precipitate has no effect on the result of the detection step and may be ignored when adding the sample e When adding samples to the AT do not remove the AT from the thermomixer to prevent the buffer from cooling down Add the sample directly into the Detection Buffer of the AT by pipetting up and down 8 Close the lids of the ATs properly to prevent them from drying out and incubate the tubes for 30 minutes at 50 C 400 rpm 9 After 30 minutes replace the Detection Buffer with 300 ul Blocking Buffer do this with one AT at a time Remove the AT from the thermomixer discard the Detection Buffer using a pipette and immediately replace with 300 ul Blocking Buffer using a new pipette tip Place the AT back in the thermomixer at 50 C and proceed with the next AT until all the AT solutions have been replaced with Blocking Buffer Incubate for 5 minutes at 50 C 400 rpm Optional e Evaporated water condensed in the lid of the AT may be removed with a pipette e g before removing the Detection Buffer containing the sample Note e Collect
3. Array Selection d Enter the lot number of the kit in the appropriate field and the name of the operator followed by pressing the Next Step button e Insert the sample codes in the screen Sample information see figure 3 Check Points 1 Array selection 2 Sample information 3 Detection step 4 Results 2 Sample information Check MDR Please enter your sample codes in the table below Figure 3 Sample information Note e Additional remarks may be added per sample optional when sample references have been filled out Double click on one of the sample reference fields A pop up Check MDR CT103 User manual Version 2 2 Issued 1 October 2012 9 CT 103 will appear see figure 4 allowing remarks to be added to individual or all samples by marking the box es of the sample s in question Check Points 1 Array selection 2 Sample information 3 Detection step 4 Results 2 Sample information Check MDR CT 103 Please enter your sample codes in the table below Sample information Aditional sample information 4 A Select all Deselect all Figure 4 Pop up for additional sample information f Goto the Detection step screen see figure 5 by clicking the Next Step button g The software will now indicate the samples to be analyzed first Check Points 1 Array selection 2 Sample information 3 Detection step 4 Results
4. User manual Check MDR CT103 Rapid Molecular Detection and Identification of Carbapenemase AmpC and ESBL genes 10 0021 V 24 021 01 Check MDR CT103 User manual Version 2 2 Issued 1 October 2012 e Check MDR CT 103 Key to symbols used For Vitro Diagnostic Use Catalog number Batch code Instructions for use number Use before YYYY MM i Consult instructions for use Temperature limitation V Contains sufficient for n tests Manufacturer INTENDED USE UE Na eO PRSE E FU ER M POSEE 2 lapiebblege m c MA 2 PRINCIPLE OF THE METHOD isse bi Rex aA 3 KIT CONTENTS FOR 24 SAMPLES i c rasa EROS 3 SHELF LIFE STORAGE AND non 4 MATERIALS REQUIRED BUT NOT SUPPLIED WITH THE KIT 4 GOOD LABORATORY PRACTICES eoa SUAVE SENATE E S 4 PROTOCOL Fm M M 5 1 DNA RECOGNITION STEP rc 5 2 DNA AMPLIFICATION STEP B 6 SP Me TEP eae E A E E N E 6 FREQUENTLY ASKED QUESTIONS FAQ 8 TROUBLESHOOTING 10 APPENDIX 1 m E T E 12 APPENDIX
5. and B the Check Points Tube Reader Important points before starting e detection step is carried out in the post PCR room e The ArrayTube DNA microarray platform see figure 1 is sold under license from Alere Technologies GmbH Procedure 1 Start preparing the required number of CP Array Tubes ATs for detection approximately 10 minutes before the end of step B One AT is required for every reaction tube Remove the ATs from their package s and place them in the thermomixer 2 Add 300 ul Detection Buffer to each AT Switch on the thermomixer and heat to 50 C It is not necessary to close the tubes Note e Be careful when removing or adding liquids with the pipette to or from the AT Do not touch the microarray at the bottom of the tube at any time Pipette all material in or out of the AT at the side of the bottom of the tube without touching the array e At 50 C remove and add solutions to one AT at a time to prevent the ATs from drying out 3 When the thermomixer has reached 50 C incubate the tubes for 2 minutes 400 rpm 4 Remove the Detection Buffer from the ATs and repeat step 3 5 Replace the Detection Buffer with 300 ul fresh Detection Buffer 6 Take the samples from step B Samples stored for longer than 2 hours after step B was completed should be heated in the PCR thermocycler at 98 C for 2 minutes CP Melt program of the PCR thermocycler see Appendix 2 Briefly spin down the reaction mixture
6. a new window with the summary of the results will appear see figure 7 which may also be printed click on the Print results button The results summary will display the sample ID and the presence absence of carbapenemases plasmidic AmpC and ESBL as well as combinations thereof see table 2 for a list of the genes that can be reported Please note that for the CTX M 1 group additional information regarding the subgroup will be displayed see Appendix 1 Click on the Quit button to end this run of analyses Check Points 1 Array selection 2 Sample information 4 Results Sample name EH CPTube 25843001 pod 34 7 sooo 1 v oaa Check MDR CT 103 Check MDR CT 103 Typing CARBA ESBL CTX M group TEM SNP WT CPTube25843003 8 1 5000003 mE ennon a 3 s needed please double click on sample name to send picture to Check Points Figure 7 Summary of the results Table 2 6 lactamase genes reported by the Check Points CTX M 1 group CTX M 1 like CTX M 15 like CTX M 3 like CTX M 32 like CTX M 2 group CTX M 8 amp 25 group CTX M 9 group Note software TEM wt TEM 104K TEM 1645 TEM 164C TEM 164H TEM 2385 SHV wt SHV 2385 SHV 238A SHV 240K WT 1645 CMY ACT MIR CMY Il Check MDR CT103
7. the liquids removed from the ATs in a disposable tube and dispose of it the same day with the other laboratory waste 10 Replace the Blocking Buffer with 300 ul fresh Blocking Buffer Set the temperature of the thermomixer to 30 C and incubate for 10 minutes 400 rpm During this time the thermomixer containing the ATs will cool down from 50 C to 30 C Check MDR CT103 User manual Version 2 2 Issued 1 October 2012 one Check MDR CT 103 11 During step 10 prepare a dilution of the Conjugate Solution black 2 ml tube with black cap with Detection Buffer using Appendix 3 at the back of this protocol For this purpose a 1 5 ml tube or a 10 ml tube may be used depending on the amount required Dispense the Conjugate Solution in the Detection Buffer by pipetting up and down 3 times Mix well by vortexing for 30 seconds Note e Conjugate Solution is stored at 20 C but is not frozen and be used directly e Conjugate dilutions have to be made fresh and should be used on the day of preparation e At30 C remove solutions from all ATs before proceeding with adding new solutions to the ATs 12 Remove the Blocking Buffer completely from all ATs Then add 150 ul conjugate dilution to each AT Incubate for 15 minutes at 30 C 400 rpm 13 Remove the conjugate dilution from the ATs and add 600 ul Detection Buffer Incubate the tubes for 2 minutes at 30 C 400 rpm 14 Replace the Detection Buffer with 600 ul fre
8. 3 Detection step Reader found 000041 Check MDR 05 Please enter CPTube LOT number place tube in reader and Confirm Sample s 5000001 CPTube LOT number m Live 5000001 No result Figure 5 Detection step 17 Enter the AT lot number in the appropriate field when the 15 minutes incubation time has been completed optional use the Barcode Reader to scan the AT lot numbers Next insert the AT with open cap into the reader close the lid of the reader and click on the Confirm button in the software Then click on Scan image the results will be 8 displayed immediately see figure 6 Finally click on Save Results to save the results in the database The software will now indicate which sample should be analyzed next Repeat this step until all ATs are analyzed Check Points 1 Array selection 2 Sample information Scan ready 3 Detection step e Reader found test o Chec k M D R 103 Please enter CPTube LOT number place tube in reader and Confirm Sample s 5000001 CPTube LOT number 25843001 Figure 6 Presentation of the final result Note e It is important to adhere to the 15 minutes incubation time with Staining Solution as much as possible step 15 A shorter incubation time may lead to faint images exceeding the 15 minutes incubation time may lead to overstaining In both cases incorrect results may be obtained 18 When all ATs have been analyzed
9. 50 C of the PCR product with the AT has been performed properly Causes may be 9 1 The picture is completely dark conjugate dilution was not removed properly Please refer to question 5 9 2 The reaction mixture after completion of step B was not added to the AT Please repeat detection step with new AT 9 3 Hybridisation temperature too high Please verify that the thermomixer temperature was 50 C when the ATs were hybridized 9 4 The BC mix was not prepared properly or was not added to the assay please repeat the test 10 The software indicates Reaction not OK please contact Check Points This message will be displayed in the following two scenarios A The software did not find the reaction control spots on the AT These amplification controls are used to check the performance of the assay in steps A and B Possible explanations are 10 1 The sample DNA was not added to the assay in step A 10 2 The sample DNA contains contaminants inhibiting the reactions These may originate from the growth medium or the DNA isolation method This may be remedied by diluting the DNA extract 10 fold with distilled water Repeat the test with the diluted DNA 10 3 The A mix was not added in step A Please repeat the test 10 4 The BC mix was not prepared properly or was not added to the assay please repeat the test Check MDR CT103 User manual Version 2 2 Issued 1 October 2012 11 12 13 14 15 16
10. Comments Figure 9 Check the box to send pictures directly over the internet Your email address Options Cancel Send Check MDR CT103 User manual Version 2 2 Issued 1 October 2012 one Check MDR CT 103 Frequently asked questions FAQ amp Troubleshooting 1 The thermocycler states an error in step A or B Please contact Check Points Technical Support support check points com 2 During the PCR programs step A or B sample s have partly evaporated Tubes may not have been closed properly Please restart the procedure from step A 3 Ihave left Solutions A B and or C out of the 20 C 4 F storage These reagents must be stored at 20 C 4 F for proper performance of the test The performance of the product cannot be fully guaranteed if these solutions were left out of 20 C 4 F for longer than 24 hours 4 Staining Solution turned blue after adding it to the AT Conjugate dilution was not properly removed by washing steps 13 and 14 detection step Continue incubation with Staining Solution for 15 minutes and take the image as described in the protocol If the image is too dark please refer to question 5 5 The picture of the array is very dark The conjugate dilution detection step 12 was not properly removed by washing steps 13 and 14 detection step Please replace the Staining Solution with Detection Buffer and take image immediately If the image is still too dark please repeat detectio
11. User manual Version 2 2 Issued 1 October 2012 e For support concerning results please send the result image along with the desired information to support check points com For this purpose double click on the result s in question in the result summary window A pop up will appear see figure 8 in which your comments may be added to the file g Send image to Check Points Figure 8 Send image to Check Points pop up Options Send picture directly over internet connection Cancel Save Send later Click on the Save Send later button to store the file which will have a cpfe extension on the computer and send it by e mail Alternatively if the computer has internet access check the box Send picture directly over internet connection and enter the reply e mail address followed by clicking on send In both cases feedback should be expected within two working days Support may also be requested at a later stage when the program has been closed For this purpose the Send image to Check Points pop up see figure 9 may also be accessed from the database viewer DBview shortcut that is located on the desktop Open the database by clicking on File followed by Open the database is located in C Mmages by default and scroll down to the results which require support By clicking on Save the Send image to Check Points pop up will appear just like in the result summary window
12. X M 37 CTX M 42 CTX M 54 CTX M 66 CTX M 88 Co 2 Check CTX M 32 CTX M 53 CTX M 55 CTX M 57 CTX M 69 MDR CT 103 The Check Points software reports four subgroups belonging to the CTX M 1 group CTX M 1 like CTX M 15 like CTX M 3 like and CTX M 32 like CTX M 1 group 12 Appendix 2 Step A Step Temperature Time Cycles 95 C 30sec 24x Step B Step Temperature Time Cycles es Gsm memes _ CH NN Step Melt Step Temperature Time Cycles Check MDR CT103 User manual Version 2 2 Issued 1 October 2012 Appendix 3 Pipetting scheme for BC mix N amp WD X Check MDR Pipetting scheme for conjugate dilution ul Conjugate ul Solution Det Buf 3 295 13 Limitations Check MDR CT103 uses a range of specific DNA markers to identify the presence or absence of ESBL AmpC and carbapenemase genes The test detects the presence of the ESBL genes TEM SHV and CTX M and it also detects Single Nucleotide Polymorphisms SNPs corresponding to amino acid positions 104 164 and 238 in TEM and 238 and 240 in SHV The nature of the above SNPs in TEM and SHV determines the ESBL phenotype ESBL yes or no for the majority of TEM and SHV types For a detailed explanation see www lahey org studies and M Gniadkowski Clin Microbiol Infect 2008 14 Suppl 1 11 32 Check MDR CT103 also detects the AmpC genes CMY DHA FOX MOX ACC ACT and MIR wh
13. al sequences by point mutations leading to amino acid substitutions www lahey org studies These amino acid substitutions may extend the substrate spectrum to hydrolyze a wide range of B lactam antibiotics i e ESBL The CTX M genes originate from Kluyvera species and are presently the most prevalent ESBLs They can be divided into 5 different groups according to their amino acid sequence CTX M 1 CTX M 2 CTX M 9 CTX M 8 and CTX M 25 The AmpC cephalosporinases are able to hydrolyze almost all B lactam antibiotics including penicillins cephalosporins and monobactams Unlike ESBLs they are not inhibited by clavulanic acid Many gram negative rod bacteria carry a chromosomal copy of an AmpC gene and plasmid encoded AmpC genes have emerged from the chromosomes of these Enterobacteriaceae Species carrying a chromosomal AmpC gene include Citrobacter freundii Morganella morganii Aeromonas caviae Hafnia alvei Enterobacter spp and Aeromonas hydrophilia see table 1 Generally plasmid encoded AmpC genes give rise to significantly increased B lactamase production compared to the chromosomally located AmpC genes Hence it is useful for the clinician to be able to discriminate between plasmid and chromosomally encoded AmpC genes The presence of a plasmidic AmpC gene is indicated when detected in a species that does not have a chromosomal AmpC e g Check MDR CT103 User manual Version 2 2 Issued 1 October 2012 ene Check MDR CT 103 K
14. ble preferable filter tips for volumes of 1 to 1000 ul 1 5 ml tubes Eppendorf tubes 10 ml tubes contact your local representative for specifications Post PCR Thermocycler Vortex Mixer Mini centrifuge Thermomixer with active cooling Check Points Tube Reader with E Ads software Computer with USB drive and internet connection Barcode Reader optional Disposable laboratory powder free gloves Pipettes amp disposable preferable filter tips for volumes of 1 to 1000 ul 1 5 ml tubes Eppendorf tubes 10 ml tubes Check MDR CT103 User manual Version 2 2 Issued 1 October 2012 ose Check MDR CT 103 Good laboratory practices Recommendations for best results The quality of the results depends on strict compliance with the following good laboratory practices especially concerning PCR e test must be performed by adequately trained personnel e Spinning down for a few seconds is done in the various steps to ensure that all material is collected at the bottom of the tubes e use reagents after their expiration date e Before use thaw frozen reagents completely at room temperature and vortex briefly to obtain a homogeneous solution After vortexing briefly spin down the solution to avoid contamination when opening the lid Avoid unnecessary freeze thawing of the kit content e Periodically verify the accuracy and precision of pipettes as well as correct functioning of th
15. ds are recommended e Store DNA extracts according to the manufacturer s protocol for the DNA extraction method used Procedure 1 Thaw all reagents i e Solution A DNA samples if kept at 20 C mix well and keep on ice 2 Forx number of samples add the following to a 1 5 ml tube x 1 2 5 ul Solution P green and x 1 5 ul Solution A brown cap Mix well and spin down briefly The solution should have a uniform blue color 3 Add 7 5 ul of this mixture to each reaction tube Next add 10 ul DNA solution of each sample NB use 1 100 part of the elution volume of the isolation and add up to 10 ul with MQ water or 0 1 TE buffer Please write down the sample reference for each tube 4 Close the tubes mix well by tapping against each tube and spin down briefly using the mini centrifuge 5 Place the tube s in the thermocycler and run the CP step A progam total sample volume 18 ul The program will run for approximately 2 5 hours The step A program is outlined in Appendix 2 5 Note e The reaction tubes NOT supplied with the kit e When closing the tube s don t use excessive pressure as the cap may distort and the sample may then evaporate during steps A and B e Refer to Appendix 2 if the settings of the thermocycler are lost 2 DNA amplification step B Important point before starting e The preparation of the reaction mix for step B is carried out the pre PCR room Procedu
16. e instruments Prevention of contaminations PCR produces a very high quantity of DNA amplification products amplicons even from minute quantities of starting material Check MDR CT103 may therefore yield unreliable results if samples become contaminated with amplicons from previous amplification reactions prior to the PCR step B of the protocol Preventive measures to minimize the risk of amplicon contamination must be taken Please read carefully and follow the instructions outlined below Use separate rooms a pre PCR room and a post PCR room e Sample preparation DNA recognition step A and preparation of the amplification step step B is carried out in the pre PCR room e Incubation in the PCR thermocycler of step B is carried out in the post PCR room e detection step is also carried out in the post PCR room Alternatively the detection step is carried out in a third room separate from the other two rooms e Never bring the reaction products of step to the pre PCR room e Never prepare the ligation and or amplification steps the post PCR room To keep laboratory free of PCR product contamination Use pipettes with hydrophobic filter tips Make sure to always use a new pipette tip when adding solutions or samples to a reaction tube to avoid contamination Follow proper pipette dispensing techniques to prevent aerosols Use separate equipment pipettes thermocyclers sample holders lab coats gloves disp
17. es it is possible that certain sub types might not be detected The test reflects the state of knowledge of Check Points Health B V The quality of the input DNA is an important factor in obtaining reliable results from Check MDR 103 DNA must be extracted from cultured bacteria using a suitable method such as commercially available extraction methods based on columns or beads Methods should be used that extract and purify total DNA i e genomic as well as plasmid DNA The presence of multiple bacterial species in a sample may hamper the interpretation of the test As with other diagnostic assays the results of this test may only be interpreted in combination with additional laboratory and clinical data available to the responsible person Use of this assay is limited to appropriately qualified personnel well trained in the test procedure and familiar with molecular biological methods The Check Points software is continuously improving thanks to the expanding global database of available results In order to further improve the software original result images including specific data are shared with this database through a default setting in the software by means of an encrypted file This data will be used solely for software improvements All information will be kept strictly confidential and will not be shared with any third parties The specific data included are the number identifying the CP Array Tube the kit lot number the software ve
18. fr N 13 APPENDIX S P 13 AN Ve c P 14 1 Intended use Check MDR CT103 is designed for the rapid molecular detection of a wide range of clinically important B lactamase genes in gram negative bacteria These include the following extended spectrum B lactamase ESBL genes AmpC genes and carbapenemase genes TEM SHV CTX M CMY DHA FOX MOX ACC MIR ACT KPC NDM OXA 48 VIM and IMP In most cases the presence of the genes indicates the expression of extended or full spectrum B lactamase activity For TEM and SHV ESBL and non ESBL variants exist and the test employs highly specific DNA markers to allow distinction between these variants The test also classifies the various CTX M genes in a number of families and subclasses Check MDR CT103 generates definitive results within the same working day compared to the 24 hours necessary for analysis with conventional phenotypic methods Information on gene type yields extra information for outbreak management Introduction The TEM SHV and CTX M genes encode the clinically most prevalent extended spectrum B lactamases ESBLs These three groups of ESBLs are generally capable of hydrolyzing first second third and fourth generation cephalosporins penicillins and monobactams thereby limiting treatment options TEM and SHV ESBL subtypes are derived from their parent
19. ich presently represent the clinically most prevalent AmpC genes It should be noted that other AmpC genes are not detected Finally Check MDR CT103 detects the carbapenemase genes KPC NDM OXA 48 VIM and IMP which presently represent the clinically most prevalent carbapenemase genes It should be noted that other carbapenemase genes are not detected Check MDR CT103 requires DNA purified from a colony or bacterial culture Clinical specimens cannot be tested directly The assay has been tested extensively with purified DNA from gram negative bacteria such as Escherichia Salmonella Klebsiella Enterobacter Citrobacter and Pseudomonas with excellent results However it may never be excluded that other gram negative bacteria or certain strains of the above species will yield poor results Furthermore although TEM SHV and CTX M are the most prevalent ESBL genes various minor ESBL genes exist However these are not frequently found in clinical settings For more details see T Naas L Poirel and P Nordmann Clin Microbiol Infect 2008 14 Suppl 1 42 52 Check MDR CT103 cannot and does not make any representation or warranty that it is capable of correctly detecting the ESBL AmpC and carbapenemase genes in all gram negative species subspecies or type or in any clinical sample source Results may need to be confirmed by additional methodologies in specific cases e g for regulatory samples Due to the high variability of bacterial genom
20. lebsiella pneumoniae or when the genotype of the AmpC gene differs from the inherent chromosomal gene of the species For example Morganella morganii contains a chromosomal copy of the DHA family Detection of this AmpC in a species other than M morganii indicates the presence of plasmid mediated AmpC Table 1 AmpC genes and the species from which they originate AmpC gene Origin of chromosomal gene Bacteria carrying ESBL or AmpC genes generally stay susceptible to carbapenem antibiotics During the last decade various carbapenemase genes have been reported associated with elevated or complete resistance against carbapenem antibiotics Strains carrying such genes are generally resistant to all B lactam antibiotics They often have additional B lactamase genes and genes for resistance against quinolones and aminoglycosides leaving very few therapeutic options The carbapenemase genes tested for with Check MDR CT103 are briefly described in the following text Klebsiella Pneumoniae Carbapenemases KPC are plasmid encoded Ambler class A carbapenemases that were first identified in Klebsiella pneumoniae isolates in North America Currently these enzymes have spread to many other parts of the world and are found in several other Enterobacteriaceae species including Escherichia coli New Delhi Metallo B lactamase NDM is a new type of carbapenemase that was first reported in 2009 when found in a Klebsiella pneumoniae and Escherichia coli
21. n step with new AT 6 Software indicates Background intensity too high See question 5 7 Thesoftware indicates Image too weak The intensity of the spots is too low Causes may be 7 1 The conjugate dilution was not prepared and or added properly Please repeat detection step with new AT 7 2 Incubation time with the Staining Solution was shorter than 15 minutes Continue incubation up to 15 minutes 7 3 Detection Buffer was not removed properly before adding Staining Solution Please repeat detection step with new AT 8 The software indicates Reference spots found The software did not find the reference spots on the AT Causes may be 10 8 1 An air bubble is interfering with the result Tap the AT gently or pipette the liquid gently up and down and then retry to take the image 8 2 The picture is very dark conjugate dilution was not removed properly Please refer to question 5 8 3 The picture is completely white Staining Solution was not added Please add Staining Solution again and proceed from detection step 15 If the results do not improve then the staining has failed Most likely no conjugate dilution was added or the conjugate dilution was not prepared properly Please repeat detection step with new AT The software indicates Hybridisation not OK The software did not find the hybridisation control spots on the AT The hybridisation control is used to check if the hybridisation at
22. o overstaining Inspect image if spots are very dark please repeat detection step with a new AT Duplicate DNA samples tested with Check MDR CT103 do not yield identical results 15 1 Inspect images for dust particles Tap or gently shake the tubes to try to remove the particles from the array and rescan the images 15 2 In case of extra spots repeat the test to confirm result May the assay be interrupted and continued at a later stage Reaction mixtures from Step A can be kept at hold at 4 C for a maximum period of two hours Reaction mixtures from Step B can be kept at 20 C for a maximum period of two weeks For further inquiries please contact Check Points Technical Support support check points com 11 17 The spot intensities on various ATs are weak Please repeat the test Contact Check Points Technical Support at support check points com if results do not improve 18 The AT image contains dust particles The software will correct this in most cases To prevent any interference with the results please take the AT out of the reader and shake it gently until the dust particles have moved to the side of the AT Check MDR CT103 User manual Version 2 2 Issued 1 October 2012 Appendix 1 CTX M 1 CTX M 23 CTX M 52 CTX M 58 CTX M 60 CTX M 61 CTX M 62 CTX M 68 CTX M 15 CTX M 28 CTX M 29 CTX M 33 CTX M 71 CTX M 72 CTX M 96 CTX M 3 CTX M 10 CTX M 11 CTX M 12 CTX M 22 CTX M 30 CTX M 34 CTX M 36 CT
23. onal methods for detection of B lactamases rely on phenotypic identification which is time consuming frequently inconclusive and not applicable to all species Therapeutic failures associated with infections caused by bacteria containing various B lactamase genes are often due to serious problems with interpretation of phenotypic tests Check MDR 103 is a rapid molecular test that detects the presence of the indicated B lactamase genes reliably predicting the expression of extended or full spectrum B lactamases Check MDR CT103 User manual Version 2 2 Issued 1 October 2012 ose Check MDR CT 103 Principle of the method The principle of the Check Points diagnostic system is based on specific molecular recognition of DNA target sequences and subsequent amplification with universal primers Each individual DNA target is recognized by a specific probe that contains a unique ZIP code corresponding to a unique position address on the microarray These ZIP codes are used for detection on the microarray after amplification Probes consist of two segments probe arms which are joined by a DNA ligase when they match perfectly with the target DNA Only connected probe arms will result in amplification products Probes that differ from the target DNA will not give amplification products even in the case of a single nucleotide difference Amplification products are hybridized to the microarray and visualized by colorimetric detection The mic
24. osables and reagents that are assigned to these rooms Never transfer items from the post PCR room to the pre PCR room Wear a clean lab coat and clean gloves during all steps of the test Wear clean gloves and a clean lab coat not previously worn while handling amplified PCR products or during sample preparation Change gloves whenever you suspect that they are contaminated Keep the tubes of all kit components and samples closed as much as possible Clean the lab benches and all equipment regularly with a 0 596 sodium hypochlorite solution Check MDR CT103 User manual Version 2 2 Issued 1 October 2012 ose Check MDR CT 103 Protocol It is strongly recommended that the full protocol is read before using the test The protocol consists of the following steps 1 DNA recognition step A 2 DNAamplification step B 3 Detection step 1 DNA recognition step A Important points before starting e Step Ais completely carried out in the pre PCR room e To ensure the best results we recommend using purified nucleic acids isolated from colonies or cells grown in nutrient broth Methods should be used that purify total DNA i e genomic as well as plasmid DNA Generally such methods copurify DNA and RNA from bacterial cells A nucleic acids concentration of 5 50 ng ul should be used compensating for the excess of RNA generally present in DNA preparations using commercially available extraction methods Column based isolation metho
25. re 1 2 Briefly spin down the reaction tubes when step A has been completed For amplification step B prepare BC mix in a 1 5 ml or 10 ml tube Take Solution B white cap O from the freezer Thaw properly at room temperature mix well and spin down briefly before use Then take Solution C red cap 9 from the freezer Prepare the required amount of BC mix using the pipetting scheme in Appendix 3 First add the required amount of Solution B to the tube Then dispense Solution C in Solution B by pipetting up and down 3 times Mix very well by vortexing and spin down briefly Add 30 ul of the freshly prepared BC mix to each reaction tube Close the tubes mix by tapping each tube and spin down briefly Transfer the tubes to the post PCR room Place the tube s in the PCR thermocycler and run the CP step B program total sample volume 48 ul The program will run for approximately 1 5 hours The step B program is outlined in Appendix 2 Briefly spin down the reaction mixture after step B has been completed Leave the reaction tubes in the PCR thermocycler at 4 C for a maximum period of two hours until step 5 of the detection step has been completed Alternatively store the reaction mixtures at 20 C for a maximum period of two weeks Check MDR CT103 User manual Version 2 2 Issued 1 October 2012 ose Check MDR CT 103 3 Detection step 1 amp mut poin ner te Che A B Figure 1 A the CP Array Tube AT
26. roarrays are contained in so called CP Array Tubes which are inserted in the Check Points Tube Reader upon completion of the detection reaction This generates an array image that is analyzed by dedicated software to yield a definitive and objective assay result Kit contents for 24 samples Components Mat No Storage conditions Description Box Room Temperature Detection Buffer 9 0007 1 bottle 80 ml Blocking Buffer 9 0008 1 bottle 20 mi Staining Solution 9 0014 1 bottle 5 ml ine due store in the dark Box 20 C Positive and negative controls are built into the system It is however strongly recommended to use a positive and negative control for each series of reactions Shelf life Storage and Handling Please check the individual components for optimal storage conditions immediately after delivery of the kit and store components accordingly Reagents stored at the appropriate storage conditions can be used until the expiration date indicated on the boxes Please visually inspect the boxes upon initial opening to ensure that their content is intact Do not use when damaged Please contact the Check Points office at support check points com if you have any questions or in case shipping has taken more than 2 days Materials required but not supplied with the kit Equipment Supplies Pre PCR Thermocycler Vortex Mixer Mini centrifuge Disposable laboratory powder free gloves Pipettes amp disposa
27. rsion used the operator and the customer name All patient data and sample information is removed in the encrypted file Check MDR CT103 User manual Version 2 2 Issued 1 October 2012 ose Check MDR CT 103 Despite the utmost care in the development and preparation of the protocol Check Points cannot take any responsibility for errors omissions and or future changes herein Literature Citation When describing a procedure for publication using this product please refer to it as the Check MDR CT103 Check Points rapid molecular detection Tel 31 317 453 908 Fax 31 317 210 147 info check points com www check points com Check Points Health BV Binnenhaven 5 6709 PD Wageningen The Netherlands 14
28. sh Detection Buffer and incubate the tubes again for 2 minutes at 30 C 400 rpm 15 Remove the Detection Buffer from the ATs and add 150 ul Staining Solution to each AT Incubate for 15 minutes at room temperature outside the thermomixer to complete the staining procedure Continue with the image analysis immediately after the 15 minutes incubation time Do not incubate with Staining Solution for more than 15 minutes because images may become too dark During the 15 minutes incubation time complete the required sample information and relevant test data in the Check Points software as outlined in point 16 a g Note e Store the bottle with Staining Solution in the dark after use 16 Filling out the experimental data a Start the computer b Start software on the computer by double clicking the Check Points desktop icon e e o Points c Double Click on Check MDR CT103 arr in the first screen Array selection as shown in figure 2 Check Points 1 Array selection 2 Sample information 3 Detection step 4 Results 1 Array selection Check MDR CT 103 Please choose array type from the list below C amp T Salmonella arr C amp T defmition 02112011 Check MDR 101 Check MDR CT101 20 1 2012 Check MDR CT102 arr Check MDR CT102 20 1 2012 Check MDR CT103 arr Check MDR CT103 20 1 2012 Please enter lot number of the kit m Operator Figure 2
29. strain from a Swedish patient repatriated from a New Delhi hospital Recent reports indicate that it has already spread to various European countries notably the U K and that NDM may be prevalent in the Indian subcontinent OXA 48 is an OXA type class D B lactamase originating from Shewanella species It is frequently found in fermenting Enterobacteriaceae like coli and Klebsiella It seems to most prevalent in Turkey and the North African subcontinent Other OXA type 2 carbapenemases like OXA 23 and OXA 58 are generally not associated with fermenting Enterobacteriaceae but occur in non fermentors like Acinetobacter baumannii VIM carbapenemase is a metallo B lactamase first reported in a Pseudomonas aeroginosa isolate found in Verona in 1997 Since then it has transferred to F coli and K pneumoniae and many other Enterobacteriaceae Presently a total of 33 variants of the VIM gene have been reported of which the DNA sequences may differ significantly Therefore VIM should be regarded as a family of homologous gene variants The IMP group of metallo B lactamases presently consists of 38 members IMP 1 was one of the first carbapenemases to be reported and was detected in Japan in 1990 in a Pseudomonas aeruginosa isolate As with VIM the gene sequences of these IMP types may vary significantly They are spread worldwide and have been found in various Enterobacteriaceae including coli Klebsiella and Enterobacter Conventi
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